UMST, Phosphoinositide-calcium signaling in cell regulation The Unit of Molecular Signal Transduction directed by Tamas Balla investigates signal transduction pathways that mediate the actions of hormones and growth factors in mammalian cells, with special emphasis on the role of phosphoinositide-derived messengers. Phosphoinositides are a small fraction of the cellular phospholipids, but play a critical role in the regulation of many (if not all) signaling protein complexes that assemble on the surface of cellular membranes. Phosphoinositides regulate protein kinases and GTP-binding proteins, as well as membrane transporters including ion channels, thereby controlling many cellular processes such as proliferation, apoptosis and metabolism. Our group focuses on one family of enzymes, the phosphatidylinositol 4-kinases (PI4Ks) that catalyze the first committed step in phosphoinositide synthesis. Current studies are aimed at (1) understanding the function and regulation of several phosphatidylinositol (PI) 4-kinases in the control of the synthesis of hormone-sensitive phosphoinositide pools; (2) characterizing the structural features that determine the catalytic specificity and inhibitor sensitivity of PI 3- and PI 4-kinases; (3) defining the molecular basis of protein-phosphoinositide interactions via the pleckstrin homology and other domains of selected regulatory proteins; (4) developing tools to analyze inositol lipid dynamics in live cells; (5) determining the importance of the lipid-protein interactions in the activation of cellular responses by G protein-coupled receptors and receptor tyrosine kinases. Pleckstrin-homology (PH) domains are protein modules of ~ 150 amino acids with a characteristic fold, and have gained great interest due to their ability to bind phosphoinositides. PH domains are present in a large variety of signaling molecules including tyrosine- or serine-threonine kinases, guanine nucleotide exchange factors and GTPase activating proteins, phospholipases and a number of adaptor proteins. Since several types of regulatory molecules possess PH domains that specifically bind to the same phosphoinositide [i.e. PI(3,4,5)P3], we wanted to investigate whether PH domains originating from various regulatory proteins and displaying identical lipid-binding specificity would interfere with PI(3,4,5)P3-regulated cellular responses in a similar manner. Therefore, several PH domains with the ability to bind phosphatidylinositol 3,4,5,-trisphosphate [PI(3,4,5)P3] were expressed in NIH 3T3 and COS-7 cells as GFP fusion proteins to determine their effects on various cellular responses known to be activated by PI(3,4,5)P3. All of these proteins, namely the PH domains of Grp1, Akt, ARNO and Btk were found to show PDGF-stimulated and wortmannin-sensitive translocation from the cytosol to the plasma membrane, indicating their ability to recognize PI(3,4,5)P3. Remarkably, while overexpressed AktPH-GFP and BtkPH-GFP were quite potent in antagonizing the PI(3,4,5)P3-mediated activation of Akt, overexpression of the other PH domains showed no such inhibitory effect. In contrast, expression of the PH domains of Grp1 and ARNO, but not those of Akt or Btk inhibited the attachment of freshly-seeded cells to culture dishes. Activation of PLCg by EGF was also attenuated by the PH domains of Grp1, ARNO and Akt, but was significantly enhanced by the Btk PH domain. By following the kinetics of expression of the various GFP-fused PH domains over several days, the PH-domain of Akt clearly showed a lipid-binding-dependent self-elimination, consistent with its interference with the anti-apoptotic Akt signaling pathway. Moreover, NIH 3T3 cells expressing the various PH domains showed characteristic morphological changes: greatly elongated extensions in response to Grp1- and ARNO-PH, and a larger foot-print size in response to the Akt PH. These data suggest that interaction with and sequestration of PI(3,4,5)P3 is not the sole mechanism by which PH domains interact with cellular membranes, and are likely to possess structural features that allow their more specific participation in the assembly and function of specific signaling pathways. Phosphatidylinositol 4-kinases catalyze the first reaction step in the synthesis of phosphoinositides. There are four mammalian PI 4-kinases identified to date; the type-III enzymes (alpha and beta forms) have similarities to PI 3-kinases within their catalytic domains, while the type-II forms (alpha and beta) belong to a new family of lipid kinases. We have previously described the differential cellular localization of these proteins and have been seeking methods by which their reaction product, PI(4)P, could be detected in singles cells. Protein domains, including PH domains, fused to the green fluorescent protein (GFP), have been successfully used to follow localized inositol lipid changes in living cells, an area where our group has been very active. The PH domains of the oxysterol binding protein (OSBP) and the adaptor protein, FAPP1, has been shown to bind PI(4)P very specifically based on in vitro binding assays. Therefore, we investigated the usefulness of the PH domains of the two proteins to detect PI(4)P in living cells. When expressed in COS-7 cells, OSBP-PH was associated with the Golgi and with small vesicles scattered around the cytoplasm, and a small amount was also found in the plasma membrane. FAPP1-PH was more prominently confined to the Golgi, especially in live cells. Expression of both domains at high levels caused fragmentation and tubulation of the Golgi that was reminiscent of the changes caused by brefeldin A. Although both PH domains showed co-localization with PI4K type-IIIbeta over the Golgi, inhibition of the enzyme by wortmannin (10 microM, up to 30 min) did not change the distribution of either PH domains. In cells overexpressing the type-II PI 4-kinases, no co-localization of either PH domains with the enzymes was found in the endocytic compartments, and only the Golgi-localized fraction of the type-IIbeta enzyme was found in co-localization with either of the PH domains. Activation of PLC by Ca2+ ionophores caused dissociation of OSBP-PH from all membrane sites, and this was rapidly reversed after restoration of Ca2+. The plasma membrane association of OSBP-PH was significantly higher after the Ca2+ transient, and was completely prevented by 10 ?M wortmannin. These data suggest that the localization of the OSBP and FAPP1 PH domains is only partially dependent on PI(4)P and that the Golgi localization of these constructs also involves additional, probably protein-protein, interactions. However, the use of these molecular tools has allowed the identification of the type-III PI 4-kinases as the source of PI(4)P formation in the plasma membrane after Ca2+ -induced PLC activation.